Rapid identification of urokinase plasminogen activator inhibitors from Traditional Chinese Medicines based on ultrafiltration, LC–MS and in silico docking

doi:10.1016/j.jpba.2018.10.036

Journal of Pharmaceutical and Biomedical Analysis

Volume 164, 5 February 2019, Pages 241-248

https://doi.org/10.1016/j.jpba.2018.10.036 Get rights and content

Highlights

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An approach integrating ultrafiltration, LC–MS, bioassay and in silico docking was developed to screen the inhibitors of uPA.
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Seven compounds in Galla Chinensis and Sanguisorbae Radix were identified as uPA ligands.
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Pentagalloylglucose and ursolic acid were discovered as potent parent structures for uPA inhibition and the SARs were investigated.

Abstract

The urokinase plasminogen activator (uPA) is regarded as the crucial trigger for plasmin generation, which is involved in several diseases especially for neoplasm metastasis. In this study, an efficient approach integrating ultrafiltration, LC/MS, bioassay and in silico docking, was proposed for rapidly detecting uPA ligands from Traditional Chinese Medicines (TCMs). Forty-two TCMs were initially assessed, and as illustrative case studies, Galla Chinensis and Sanguisorbae Radix, which appeared significant inhibitory activities on uPA, were chosen to develpe and verify the strategy. A total of seven uPA ligands were successfully detected and identified. Two of them, pentagalloylglucose and 28-O-β-d-glucopyranosyl pomolic acid, were demonstrated to be potential inhibitors, with IC₅₀ at 1.639 μM and 37.82 μM repectively. Furthermore, a combinatorial compound library screening combined with in silico docking assay, was revealed that ursolic acid (IC₅₀ = 2.623 μM) was also speculated to be a potent parent structure for inhibition of uPA. This approach offers a multidimensional perspective to discover uPA-binding leading compounds from TCMs or other complex mixtures, which would provide an efficient route for drug discovery.

Graphical abstract

Introduction

Urokinase-type plasminogen activator (uPA, EC 3.4.21.73) is one of the two human proteases that mediate the proteolytic cleavage of plasminogen into plasmin [1,2]. uPA is overexpressed in several malignant tumors such as breast, prostate and bladder cancer [[3], [4], [5]], and it has been served as a prognostic marker in cancer [6,7]. Tumor cells or the adjacent stroma secrete uPA, and then uPA can bound to the cell surface receptor, uPAR [8]. uPA–uPAR interaction can initiate both ECM degradation and signal transduction, which in turn facilitates the invasion and metastasis of tumor cells [9]. Inhibition of uPA has been deemed as a therapeutic approach for uPA-dependent tumor metastasis [[10], [11], [12]]. Amiloride, an available effective uPA inhibitor [13], was reported to suppress metastasis of colon and breast tumors [[14], [15], [16]], whereas side effects such as gastrointestinal symptoms [17] were accompanied.

Traditional Chinese Medicines (TCMs) have served as an invaluable source for discovering bioactive compounds [18,19]. Natural uPA inhibitors from TCMs have been explored as viable alternatives for the treatment of tumor metastasis or other uPA-related diseases. In this study, forty-two widely used TCMs had been screened for their inhibitory activities against uPA. Among them, Galla Chinensis and Sanguisorbae Radix showed high inhibitory activities against uPA, which were consistent with the previous reports [20] and traditional uses [21]. However, the specific bioactive compounds involved remained elusive.

The conventional multiple-step isolation method for screening bioactive compounds from TCMs is time-consuming and labor-intensive [22]. To enhance the throughput of drug discovery, several methods based on protein–ligand interaction have been developed. Therein, the method integrating ultrafiltration and LC–MS (UF-LC–MS) has been proven to be a powerful tool for rapidly screening and identifying affinity ligands of macromolecules from complex mixtures [23,24]. The general process of UF-LC–MS consists of three steps: incubation, ultrafiltration, and characterization. After incubation, the ultrafiltration separates the ligand-receptor complexes from the unbound compounds. Then the ligands could be released from the complexes and identified by HPLC-MS/MS analysis. This method is time-saving, cost-effective and convenient, which has obtained increasing attention and applied on screening natural inhibitors of various enzymes such as α-glucosidase [[25], [26], [27]], human serum albumin [28] and xanthine oxidase [29]. However, to our best knowledge, UF-LC–MS method has not been developed for finding uPA ligands from TCMs or other complex mixtures.

Moreover, to increase the chance of encountering small molecules with excellent activities, a lead-derived combinatorial compound library [24] was also used for uPA inhibitors screening. Derivatives of lead compounds would be obtained by elimination, dissociation, transposition or introduction of some chemical constitution and formed the combinational library, in which more potent inhibitors may occur. Meanwhile, structure-activity relationships (SARs) could be revealed preliminarily.

This study was initially designed to develop an approach integrating UF-LC–MS and enzyme activity assay for screening uPA inhibitors from TCMs. To increase the capture efficiency of fishing bioactive compounds, two TCMs including Galla Chinensis and Sanguisorbae Radix, which have higher inhibitory activities to uPA, were selected as examples. A total of seven uPA ligands were identified by UF-LC–MS, and two of them, pentagalloylglucose and 28-O-β-D-glucopyranosyl pomolic acid, were proven as potent uPA inhibitors. Moreover, to extend the searching scope and screen potent inhibitors efficiently, an additional screening with a combinatorial compound library based on pentagalloylglucose and 28-O-β-D-glucopyranosyl pomolic acid was conducted. Herein, ursolic acid was also identified as a potent inhibitor and the mechanism of interaction was investigated by in silico docking. The present strategy is expected to accelerate the natural-product based drug discovery.

Section snippets

Materials, chemicals, and reagents

The Galla Chinensis, Sanguisorbae Radix and other TCMs for screening were purchased from Tong Ren Tang Industrial Corporation (Nanjing, China). The samples were dried at 60 °C for 3 h, and powdered, then stored in a refrigerator at 4 °C. Voucher specimens were identificated and deposited in Jiangsu Key Laboratory of TCM Evaluation and Translational Research, China Pharmaceutical University.

Standards of gallic acid, methyl gallate, ziyuglycoside I, ellagic acid, ursolic acid, oleanolic acid,

Strategy for screening uPA inhibitors from TCMs

The strategy for screening uPA inhibitors from TCMs is summarized in Fig. 1. Firstly, the preliminary screening is conducted. In this step, several TCMs associated with uPA-related diseases would be initially assessed for their inhibitory activities on uPA. To narrow down the scope and select appropriate examples for screening, TCMs with higher inhibitory activities on uPA would be filtered out and chosen for screening potential uPA inhibitors. The second step is to reveal the scientific basis

Conclusions

An effective strategy integrating UF-LC–MS, bioassay and in silico docking was proposed for screening candidate inhibitors of uPA from TCMs. After preliminary screening, Galla Chinensis and Sanguisorbae Radix, which were identified with high inhibitory activity, were selected as illustrations for uPA inhibitors screening. According to UF-LC–MS method, a total of seven uPA ligands were detected and identified, in which pentagalloylglucose and 28-O-β-D-glucopyranosyl pomolic acid were selected

Acknowledgements

This study was supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and Natural Science Foundation of Jiangsu Province (BK20160756).

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